RESUMO
ß3-Adrenergic receptor (ß3AR) agonists have been shown to protect against ischemia-reperfusion injury (IRI). Since ß3ARs are present both in cardiomyocytes and in endothelial cells, the cellular compartment responsible for this protection has remained unknown. Using transgenic mice constitutively expressing the human ß3AR (hß3AR) in cardiomyocytes or in the endothelium on a genetic background of null endogenous ß3AR expression, we show that only cardiomyocyte expression protects against IRI (45 min ischemia followed by reperfusion over 24 h). Infarct size was also limited after ischemia-reperfusion in mice with cardiomyocyte hß3AR overexpression on top of endogenous ß3AR expression. hß3AR overexpression in these mice reduced IRI-induced cardiac fibrosis and improved long-term left ventricular systolic function. Cardiomyocyte-specific ß3AR overexpression resulted in a baseline remodeling of the mitochondrial network, characterized by upregulated mitochondrial biogenesis and a downregulation of mitochondrial quality control (mitophagy), resulting in elevated numbers of small mitochondria with a depressed capacity for the generation of reactive oxygen species but improved capacity for ATP generation. These processes precondition cardiomyocyte mitochondria to be more resistant to IRI. Upon reperfusion, hearts with hß3AR overexpression display a restoration in the mitochondrial quality control and a rapid activation of antioxidant responses. Strong protection against IRI was also observed in mice infected with an adeno-associated virus (AAV) encoding hß3AR under a cardiomyocyte-specific promoter. These results confirm the translational potential of increased cardiomyocyte ß3AR expression, achieved either naturally through exercise or artificially through gene therapy approaches, to precondition the cardiomyocyte mitochondrial network to withstand future insults.
RESUMO
BACKGROUND: Cardiac ventricles provide the contractile force of the beating heart throughout life. How the primitive endocardium-layered myocardial projections called trabeculae form and mature into the adult ventricles is of great interest for biology and regenerative medicine. Trabeculation is dependent on the signaling protein Nrg1 (neuregulin-1). However, the mechanism of action of Nrg1 and its role in ventricular wall maturation are poorly understood. METHODS: We investigated the functions and downstream mechanisms of Nrg1 signaling during ventricular chamber development using confocal imaging, transcriptomics, and biochemical approaches in mice with cardiac-specific inactivation or overexpression of Nrg1. RESULTS: Analysis of cardiac-specific Nrg1 mutant mice showed that the transcriptional program underlying cardiomyocyte-oriented cell division and trabeculae formation depends on endocardial Nrg1 to myocardial ErbB2 (erb-b2 receptor tyrosine kinase 2) signaling and phospho-Erk (phosphorylated extracellular signal-regulated kinase; pErk) activation. Early endothelial loss of Nrg1 and reduced pErk activation diminished cardiomyocyte Pard3 and Crumbs2 (Crumbs Cell Polarity Complex Component 2) protein and altered cytoskeletal gene expression and organization. These alterations are associated with abnormal gene expression related to mitotic spindle organization and a shift in cardiomyocyte division orientation. Nrg1 is crucial for trabecular growth and ventricular wall thickening by regulating an epithelial-to-mesenchymal transition-like process in cardiomyocytes involving migration, adhesion, cytoskeletal actin turnover, and timely progression through the cell cycle G2/M phase. Ectopic cardiac Nrg1 overexpression and high pErk signaling caused S-phase arrest, sustained high epithelial-to-mesenchymal transition-like gene expression, and prolonged trabeculation, blocking compact myocardium maturation. Myocardial trabecular patterning alterations resulting from above- or below-normal Nrg1-dependent pErk activation were concomitant with sarcomere actin cytoskeleton disorganization. The Nrg1 loss- and gain-of-function transcriptomes were enriched for Yap1 (yes-associated protein-1) gene signatures, identifying Yap1 as a potential downstream effector. Furthermore, biochemical and imaging data reveal that Nrg1 influences pErk activation and Yap1 nuclear-cytoplasmic distribution during trabeculation. CONCLUSIONS: These data establish the Nrg1-ErbB2/ErbB4-Erk axis as a crucial regulator of cardiomyocyte cell cycle progression and migration during ventricular development.
Assuntos
Miócitos Cardíacos , Neuregulina-1 , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Neuregulina-1/genética , Miocárdio/metabolismo , Ventrículos do Coração/metabolismo , Divisão CelularRESUMO
Aortic stenosis (AS) is associated with left ventricular (LV) hypertrophy and heart failure (HF). There is a lack of therapies able to prevent/revert AS-induced HF. Beta3 adrenergic receptor (ß3AR) signaling is beneficial in several forms of HF. Here, we studied the potential beneficial effect of ß3AR overexpression on AS-induced HF. Selective ß3AR stimulation had a positive inotropic effect. Transgenic mice constitutively overexpressing human ß3AR in the heart (c-hß3tg) were protected from the development of HF in response to induced AS, and against cardiomyocyte mitochondrial dysfunction (fragmented mitochondria with remodeled cristae and metabolic reprogramming featuring altered substrate use). Similar beneficial effects were observed in wild-type mice inoculated with adeno-associated virus (AAV9) inducing cardiac-specific overexpression of human ß3AR before AS induction. Moreover, AAV9-hß3AR injection into wild-type mice at late disease stages, when cardiac hypertrophy and metabolic reprogramming are already advanced, reversed the HF phenotype and restored balanced mitochondrial dynamics, demonstrating the potential of gene-therapy-mediated ß3AR overexpression in AS. Mice with cardiac specific ablation of Yme1l (cYKO), characterized by fragmented mitochondria, showed an increased mortality upon AS challenge. AAV9-hß3AR injection in these mice before AS induction reverted the fragmented mitochondria phenotype and rescued them from death. In conclusion, our results step out that ß3AR overexpression might have translational potential as a therapeutic strategy in AS-induced HF.
Assuntos
Estenose da Valva Aórtica , Insuficiência Cardíaca , Humanos , Camundongos , Animais , Receptores Adrenérgicos beta 3 , Dinâmica Mitocondrial , Hipertrofia Ventricular Esquerda , Miócitos Cardíacos , Camundongos Transgênicos , MetaloendopeptidasesRESUMO
Bone morphogenetic protein (Bmp) signaling is critical for organismal development and homeostasis. To elucidate Bmp2 function in the vascular/hematopoietic lineages we generated a new transgenic mouse line in which ectopic Bmp2 expression is controlled by the Tie2 promoter. Tie2CRE/+;Bmp2tg/tg mice develop aortic valve dysfunction postnatally, accompanied by pre-calcific lesion formation in valve leaflets. Remarkably, Tie2CRE/+;Bmp2tg/tg mice develop extensive soft tissue bone formation typical of acquired forms of heterotopic ossification (HO) and genetic bone disorders, such as Fibrodysplasia Ossificans Progressiva (FOP). Ectopic ossification in Tie2CRE/+;Bmp2tg/tg transgenic animals is accompanied by increased bone marrow hematopoietic, fibroblast and osteoblast precursors and circulating pro-inflammatory cells. Transplanting wild-type bone marrow hematopoietic stem cells into lethally irradiated Tie2CRE/+;Bmp2tg/tg mice significantly delays HO onset but does not prevent it. Moreover, transplanting Bmp2-transgenic bone marrow into wild-type recipients does not result in HO, but hematopoietic progenitors contribute to inflammation and ectopic bone marrow colonization rather than to endochondral ossification. Conversely, aberrant Bmp2 signaling activity is associated with fibroblast accumulation, skeletal muscle fiber damage, and expansion of a Tie2+ fibro-adipogenic precursor cell population, suggesting that ectopic bone derives from a skeletal muscle resident osteoprogenitor cell origin. Thus, Tie2CRE/+;Bmp2tg/tg mice recapitulate HO pathophysiology, and might represent a useful model to investigate therapies seeking to mitigate disorders associated with aberrant extra-skeletal bone formation.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Linhagem da Célula , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/patologia , Receptor TIE-2/metabolismo , Animais , Valva Aórtica/diagnóstico por imagem , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Transplante de Medula Óssea , Proteína Morfogenética Óssea 2/sangue , Calcinose/diagnóstico por imagem , Calcinose/patologia , Calcinose/fisiopatologia , Condrogênese , Células Endoteliais/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Estimativa de Kaplan-Meier , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Musculares/patologia , Ossificação Heterotópica/sangue , Ossificação Heterotópica/diagnóstico por imagem , Osteogênese , Tomografia Computadorizada por Raios XRESUMO
Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational weeks 9, 13, and 22 and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at week 9 rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.
Assuntos
Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/genética , Valva Aórtica/patologia , Calcinose/sangue , Calcinose/genética , Doenças Fetais/genética , Transcriptoma , Adulto , Valva Aórtica/embriologia , Estenose da Valva Aórtica/embriologia , Estenose da Valva Aórtica/epidemiologia , Doenças Assintomáticas , Biomarcadores/sangue , Calcinose/embriologia , Calcinose/epidemiologia , Estudos de Casos e Controles , Análise por Conglomerados , Feminino , Idade Gestacional , Humanos , Valva Mitral/embriologia , Valva Mitral/patologia , Gravidez , Estudos Prospectivos , RNA-Seq , Espanha/epidemiologia , Valva Tricúspide/embriologia , Valva Tricúspide/patologiaRESUMO
Coronaries are essential for myocardial growth and heart function. Notch is crucial for mouse embryonic angiogenesis, but its role in coronary development remains uncertain. We show Jag1, Dll4 and activated Notch1 receptor expression in sinus venosus (SV) endocardium. Endocardial Jag1 removal blocks SV capillary sprouting, while Dll4 inactivation stimulates excessive capillary growth, suggesting that ligand antagonism regulates coronary primary plexus formation. Later endothelial ligand removal, or forced expression of Dll4 or the glycosyltransferase Mfng, blocks coronary plexus remodeling, arterial differentiation, and perivascular cell maturation. Endocardial deletion of Efnb2 phenocopies the coronary arterial defects of Notch mutants. Angiogenic rescue experiments in ventricular explants, or in primary human endothelial cells, indicate that EphrinB2 is a critical effector of antagonistic Dll4 and Jag1 functions in arterial morphogenesis. Thus, coronary arterial precursors are specified in the SV prior to primary coronary plexus formation and subsequent arterial differentiation depends on a Dll4-Jag1-EphrinB2 signaling cascade.
Assuntos
Vasos Coronários/crescimento & desenvolvimento , Vasos Coronários/metabolismo , Efrina-B2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Endocárdio/metabolismo , Endotélio Vascular/metabolismo , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipóxia/metabolismo , Hipóxia/fisiopatologia , Ligantes , Camundongos , Morfogênese , Mutação/genética , Fatores de Transcrição NFATC/metabolismo , Neovascularização Fisiológica , Receptores Notch/metabolismo , Estresse Fisiológico , Transcriptoma/genética , Remodelação VascularRESUMO
The epicardium, the outer mesothelial layer enclosing the myocardium, plays key roles in heart development and regeneration. During embryogenesis, the epicardium arises from the proepicardium (PE), a cell cluster that appears in the dorsal pericardium (DP) close to the venous pole of the heart. Little is known about how the PE emerges from the pericardial mesothelium. Using a zebrafish model and a combination of genetic tools, pharmacological agents and quantitative in vivo imaging, we reveal that a coordinated collective movement of DP cells drives PE formation. We found that Bmp signaling and the actomyosin cytoskeleton promote constriction of the DP, which enables PE cells to extrude apically. We provide evidence that cell extrusion, which has been described in the elimination of unfit cells from epithelia and the emergence of hematopoietic stem cells, is also a mechanism for PE cells to exit an organized mesothelium and fulfil their developmental fate to form a new tissue layer, the epicardium.
Assuntos
Actinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Movimento Celular , Coração/embriologia , Pericárdio/citologia , Pericárdio/embriologia , Células-Tronco/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular/genética , Embrião não Mamífero , Miocárdio/citologia , Organogênese/genética , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-ß2 (TGFß2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfß2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.
Assuntos
Endocárdio/embriologia , Endocárdio/metabolismo , Valvas Cardíacas/embriologia , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Benzazepinas/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Endocárdio/citologia , Endocárdio/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Valvas Cardíacas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Camundongos Mutantes , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores Notch/genética , Reprodutibilidade dos TestesRESUMO
Abnormalities of the arterial valve leaflets, predominantly bicuspid aortic valve, are the commonest congenital malformations. Although many studies have investigated the development of the arterial valves, it has been assumed that, as with the atrioventricular valves, endocardial to mesenchymal transition (EndMT) is the predominant mechanism. We show that arterial is distinctly different from atrioventricular valve formation. Whilst the four septal valve leaflets are dominated by NCC and EndMT-derived cells, the intercalated leaflets differentiate directly from Tnnt2-Cre+/Isl1+ progenitors in the outflow wall, via a Notch-Jag dependent mechanism. Further, when this novel group of progenitors are disrupted, development of the intercalated leaflets is disrupted, resulting in leaflet dysplasia and bicuspid valves without raphe, most commonly affecting the aortic valve. This study thus overturns the dogma that heart valves are formed principally by EndMT, identifies a new source of valve interstitial cells, and provides a novel mechanism for causation of bicuspid aortic valves without raphe.
Assuntos
Valva Aórtica/anormalidades , Células Epiteliais/patologia , Doenças das Valvas Cardíacas/patologia , Proteína Jagged-1/genética , Miócitos de Músculo Liso/patologia , Receptor Notch1/genética , Células-Tronco/patologia , Animais , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula/genética , Rastreamento de Células/métodos , Embrião de Mamíferos , Células Epiteliais/metabolismo , Expressão Gênica , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/metabolismo , Humanos , Integrases/genética , Integrases/metabolismo , Proteína Jagged-1/metabolismo , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Miócitos de Músculo Liso/metabolismo , Receptor Notch1/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Troponina T/genética , Troponina T/metabolismoRESUMO
In vertebrate hearts, the ventricular trabecular myocardium develops as a sponge-like network of cardiomyocytes that is critical for contraction and conduction, ventricular septation, papillary muscle formation and wall thickening through the process of compaction 1 . Defective trabeculation leads to embryonic lethality2-4 or non-compaction cardiomyopathy (NCC) 5 . There are divergent views on when and how trabeculation is initiated in different species. In zebrafish, trabecular cardiomyocytes extrude from compact myocardium 6 , whereas in chicks, chamber wall thickening occurs before overt trabeculation 7 . In mice, the onset of trabeculation has not been described, but is proposed to begin at embryonic day 9.0, when cardiomyocytes form radially oriented ribs 2 . Endocardium-myocardium communication is essential for trabeculation, and numerous signalling pathways have been identified, including Notch2,8 and Neuregulin (NRG) 4 . Late disruption of the Notch pathway causes NCC 5 . Whereas it has been shown that mutations in the extracellular matrix (ECM) genes Has2 and Vcan prevent the formation of trabeculae in mice9,10 and the matrix metalloprotease ADAMTS1 promotes trabecular termination 3 , the pathways involved in ECM dynamics and the molecular regulation of trabeculation during its early phases remain unexplored. Here we present a model of trabeculation in mice that integrates dynamic endocardial and myocardial cell behaviours and ECM remodelling, and reveal new epistatic relationships between the involved signalling pathways. NOTCH1 signalling promotes ECM degradation during the formation of endocardial projections that are critical for individualization of trabecular units, whereas NRG1 promotes myocardial ECM synthesis, which is necessary for trabecular rearrangement and growth. These systems interconnect through NRG1 control of Vegfa, but act antagonistically to establish trabecular architecture. These insights enabled the prediction of persistent ECM and cardiomyocyte growth in a mouse NCC model, providing new insights into the pathophysiology of congenital heart disease.
Assuntos
Coração/embriologia , Miocárdio/citologia , Miocárdio/metabolismo , Neuregulina-1/metabolismo , Organogênese , Receptor Notch1/metabolismo , Animais , Modelos Animais de Doenças , Endocárdio/citologia , Endocárdio/metabolismo , Matriz Extracelular/metabolismo , Cardiopatias/congênito , Cardiopatias/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Neuregulina-1/genética , Receptor Notch1/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Fstl1 (Follistatin-like 1) is a secreted protein that is expressed in the atrioventricular valves throughout embryonic development, postnatal maturation, and adulthood. In this study, we investigated the loss of Fstl1 in the endocardium/endothelium and their derived cells. APPROACH AND RESULTS: We conditionally ablated Fstl1 from the endocardial lineage using a transgenic Tie2-Cre mouse model. These mice showed a sustained Bmp and Tgfß signaling after birth. This resulted in ongoing proliferation and endocardial-to-mesenchymal transition and ultimately in deformed nonfunctional mitral valves and a hypertrophic dilated heart. Echocardiographic and electrocardiographic analyses revealed that loss of Fstl1 leads to mitral regurgitation and left ventricular diastolic dysfunction. Cardiac function gradually deteriorated resulting in heart failure with preserved ejection fraction and death of the mice between 2 and 4 weeks after birth. CONCLUSIONS: We report on a mouse model in which deletion of Fstl1 from the endocardial/endothelial lineage results in deformed mitral valves, which cause regurgitation, heart failure, and early cardiac death. The findings provide a potential molecular target for the clinical research into myxomatous mitral valve disease.
Assuntos
Linhagem da Célula , Endocárdio/metabolismo , Células Endoteliais/metabolismo , Proteínas Relacionadas à Folistatina/deficiência , Insuficiência Cardíaca/metabolismo , Insuficiência da Valva Mitral/metabolismo , Prolapso da Valva Mitral/metabolismo , Valva Mitral/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Endocárdio/patologia , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal , Proteínas Relacionadas à Folistatina/genética , Predisposição Genética para Doença , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Integrases/genética , Camundongos Knockout , Valva Mitral/patologia , Valva Mitral/fisiopatologia , Insuficiência da Valva Mitral/genética , Insuficiência da Valva Mitral/patologia , Insuficiência da Valva Mitral/fisiopatologia , Prolapso da Valva Mitral/genética , Prolapso da Valva Mitral/patologia , Prolapso da Valva Mitral/fisiopatologia , Fenótipo , Receptor TIE-2/genética , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda , Remodelação VentricularRESUMO
Mesenchymal stem cells (MSCs) are effective in treating several pathologies. We and others have demonstrated that hypoxia or hypoxia-inducible factor 1 alpha (HIF-1α) stabilization improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. To further explore the mechanisms induced by HIF-1α in MSCs, we studied its relationship with Notch signaling and observed that overexpression of HIF-1α in MSCs increased protein levels of the Notch ligands Jagged 1-2 and Delta-like (Dll)1, Dll3, and Dll4 and potentiated Notch signaling only when this pathway was activated. Crosstalk between HIF and Notch resulted in Notch-dependent migration and spreading of MSCs, which was abolished by γ-secretase inhibition. However, the HIF-1-induced increase in MSC proliferation was independent of Notch signaling. The ubiquitin family member, small ubiquitin-like modifier (SUMO), has important functions in many cellular processes and increased SUMO1 protein levels have been reported in hypoxia. To investigate the potential involvement of SUMOylation in HIF/Notch crosstalk, we measured general SUMOylation levels and observed increased SUMOylation in HIF-1-expressing MSCs. Moreover, proliferation and migration of MSCs were reduced in the presence of a SUMOylation inhibitor, and this effect was particularly robust in HIF-MSCs. Immunoprecipitation studies demonstrated SUMOylation of the intracellular domain of Notch1 (N1ICD) in HIF-1-expressing MSCs, which contributed to Notch pathway activation and resulted in increased levels of N1ICD nuclear translocation as assessed by subcellular fractionation. SUMOylation of N1ICD was also observed in HEK293T cells with stabilized HIF-1α expression, suggesting that this is a common mechanism in eukaryotic cells. In summary, we describe, for the first time, SUMOylation of N1ICD, which is potentiated by HIF signaling. These phenomena could be relevant for the therapeutic effects of MSCs in hypoxia or under conditions of HIF stabilization.
Assuntos
Proliferação de Células/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/metabolismo , Receptor Notch1/genética , Sumoilação/genética , Secretases da Proteína Precursora do Amiloide/genética , Hipóxia Celular/genética , Movimento Celular/genética , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ligação Proteica , Transdução de Sinais , Ubiquitina/genéticaRESUMO
RATIONALE: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood. OBJECTIVE: The aim of this study is to determine the functional specificity of Notch in valve development. METHODS AND RESULTS: Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype. CONCLUSIONS: During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.
Assuntos
Valva Mitral/metabolismo , Morfogênese , Receptor Notch1/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Valva Mitral/anormalidades , Valva Mitral/embriologia , Receptor Notch1/metabolismo , Regulação para CimaRESUMO
Congenital coronary artery anomalies are of major significance in clinical cardiology and cardiac surgery due to their association with myocardial ischaemia and sudden death. Such anomalies are detectable by imaging modalities and, according to various definitions, their prevalence ranges from 0.21 to 5.79%. This consensus document from the Development, Anatomy and Pathology Working Group of the European Society of Cardiology aims to provide: (i) a definition of normality that refers to essential anatomical and embryological features of coronary vessels, based on the integrated analysis of studies of normal and abnormal coronary embryogenesis and pathophysiology; (ii) an animal model-based systematic survey of the molecular and cellular mechanisms that regulate coronary blood vessel development; (iii) an organization of the wide spectrum of coronary artery anomalies, according to a comprehensive anatomical and embryological classification scheme; (iv) current knowledge of the pathophysiological mechanisms underlying symptoms and signs of coronary artery anomalies, with diagnostic and therapeutic implications. This document identifies the mosaic-like embryonic development of the coronary vascular system, as coronary cell types differentiate from multiple cell sources through an intricate network of molecular signals and haemodynamic cues, as the necessary framework for understanding the complex spectrum of coronary artery anomalies observed in human patients.
Assuntos
Doença da Artéria Coronariana/congênito , Anomalias dos Vasos Coronários , Vasos Coronários , Coração/anatomia & histologia , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Animais , Cardiologia/métodos , Doença da Artéria Coronariana/patologia , Anomalias dos Vasos Coronários/embriologia , Anomalias dos Vasos Coronários/patologia , Anomalias dos Vasos Coronários/fisiopatologia , Vasos Coronários/anatomia & histologia , Vasos Coronários/crescimento & desenvolvimento , Vasos Coronários/patologia , Coração/fisiologia , HumanosRESUMO
Heart valve development begins with the endothelial-to-mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate-mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock-in tamoxifen-inducible Cre line was recently generated (Msx1CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock-in allele and track the Msx1-expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling.
Assuntos
Alelos , Técnicas de Introdução de Genes , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Integrases/genética , Fator de Transcrição MSX1/genética , Receptores de Estrogênio/genética , Animais , Endocárdio/enzimologia , Endocárdio/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Integrases/metabolismo , Fator de Transcrição MSX1/metabolismo , Camundongos , Organogênese/genética , Receptores de Estrogênio/metabolismoRESUMO
NOTCH signaling suppresses tumor growth and proliferation in several types of stratified epithelia. Here, we show that missense mutations in NOTCH1 and NOTCH2 found in human bladder cancers result in loss of function. In murine models, genetic ablation of the NOTCH pathway accelerated bladder tumorigenesis and promoted the formation of squamous cell carcinomas, with areas of mesenchymal features. Using bladder cancer cells, we determined that the NOTCH pathway stabilizes the epithelial phenotype through its effector HES1 and, consequently, loss of NOTCH activity favors the process of epithelial-mesenchymal transition. Evaluation of human bladder cancer samples revealed that tumors with low levels of HES1 present mesenchymal features and are more aggressive. Together, our results indicate that NOTCH serves as a tumor suppressor in the bladder and that loss of this pathway promotes mesenchymal and invasive features.
Assuntos
Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Receptor Notch1/genética , Receptor Notch2/genética , Fatores de Transcrição HES-1 , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologiaRESUMO
The basic-helix-loop-helix (bHLH) transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here, we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardium-to-endocardium-based cell signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endocárdio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptores Notch/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Endocárdio/embriologia , Camundongos , Neuregulina-1/genética , Neuregulina-1/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Receptores Notch/genética , Ativação Transcricional , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION: Dysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells. METHODS: We have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors. RESULTS: We found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining. CONCLUSIONS: These results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Receptores Notch/metabolismo , Animais , Neoplasias da Mama/genética , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Domínios e Motivos de Interação entre Proteínas/genética , Receptor Notch1/química , Receptor Notch1/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Receptores Notch/antagonistas & inibidores , Receptores Notch/química , Transdução de Sinais/efeitos dos fármacos , Carga TumoralRESUMO
Epithelial to mesenchymal transition (EMT) is a fundamental process during development and disease, including development of the heart valves and tumour metastases. An extended cellular Potts model was implemented to represent the behaviour emerging from autonomous cell morphology, labile adhesion, junctional coupling and cell motility. Computer simulations normally focus on these functional changes independently whereas this model facilitates exploration of the interplay between cell shape changes, adhesion and migration. The simulation model is fitted to an in vitro model of endocardial EMT, and agrees with the finding that Notch signalling increases cell-matrix adhesion in addition to modulating cell-cell adhesion.
Assuntos
Adesão Celular , Transição Epitelial-Mesenquimal , Receptores Notch/metabolismo , Algoritmos , Animais , Caderinas/metabolismo , Comunicação Celular , Movimento Celular , Simulação por Computador , Endocárdio/patologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Células MCF-7 , Camundongos , Modelos Biológicos , Transdução de SinaisRESUMO
Zebrafish have the capacity to regenerate several organs, including the heart and fins. Fin regeneration is epimorphic, involving the formation at the amputation plane of a mass of undifferentiated, proliferating mesenchymal progenitor-like cells, called blastema. This tissue provides all the cell types that form the fin, so that after damage or amputation the fin pattern and structure are fully restored. How blastema cells remain in this progenitor-like state is poorly understood. Here, we show that the Notch pathway plays an essential role during fin regeneration. Notch signalling is activated during blastema formation and remains active throughout the regeneration process. Chemical inhibition or morpholino-mediated knockdown of Notch signalling impairs fin regeneration via decreased proliferation accompanied by reduced expression of Notch target genes in the blastema. Conversely, overexpression of a constitutively active form of the Notch1 receptor (N1ICD) in the regenerating fin leads to increased proliferation and to the expansion of the blastema cell markers msxe and msxb, as well as increased expression of the proliferation regulator aldh1a2. This blastema expansion prevents regenerative fin outgrowth, as indicated by the reduction in differentiating osteoblasts and the inhibition of bone regeneration. We conclude that Notch signalling maintains blastema cells in a plastic, undifferentiated and proliferative state, an essential requirement for fin regeneration.